Aims: Ero1 flavoproteins catalyse oxidative folding in the endoplasmic reticulum (ER), consuming oxygen and generating hydrogen peroxide (H2O2). The ER-located glutathione peroxidase 7 (GPx7) shows protein disulphide isomerase (PDI)-dependent peroxidase activity in vitro. Our work aims to identify the physiological role of GPx7 in the Ero1α/PDI oxidative folding pathway and dissect the reaction mechanisms of GPx7. Results: Our data show that GPx7 can utilize Ero1α-produced H2O2 to accelerate oxidative folding of substrates both in vitro and in vivo. H2O2 oxidises Cys57 of GPx7 to sulfenic acid, which can be resolved by Cys86 to form an intramolecular disulphide bond. Both the disulphide form and sulfenic acid form of GPx7 can oxidise PDI for catalysing oxidative folding. GPx7 prefers to interact with the a domain of PDI, and intramolecular cooperation between the two redox-active sites of PDI increases the activity of the Ero1α/GPx7/PDI triad. Innovation: Our in vitro and in vivo evidences provide mechanistic insights into how cells consume potentially harmful H2O2 while optimizing oxidative protein folding via the Ero1α/GPx7/PDI triad. Cys57 can promote PDI oxidation in two ways and Cys86 emerges as a novel non-canonical resolving cysteine. Conclusion: GPx7 promotes oxidative protein folding directly utilizing Ero1α-generated H2O2 in the early secretory compartment. Thus, the Ero1α/GPx7/PDI triad generates two disulphide bonds and two H2O molecules at the expense of a single O2 molecule.
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